Kidney Development And Gene Expression In The Hif2 Alpha Knockout Mouse

The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2 alpha and HIF1 beta subunits, drives expression of genes essential for vascularization, including vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2 alpha/LacZ transgenic mouse to define patterns of HIF2 alpha transcription during kidney development and maturation. Our results from embryonic heterozygotes showed HIF2 alpha/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age, glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells. These expression patterns were confirmed by electron microscopy using Bluo-gal as a beta-galactosidase substrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stages examined. Light and electron microscopic examination of kidneys from HIF2 alpha null embryos showed no defects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein were seen on Western blots of kidney extracts from homozygous and heterozygous HIF2 alpha mutants. To examine responsiveness of HIF2 alpha null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in room air or in mild (5% O-2) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled when metanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2 alpha knockouts were morphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial cell adhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1. and WT-1, were all expressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction of kidneys from HIF2 alpha null embryos and wild-type siblings and found no compensatory up-regulation of HIF1 alpha or -3 alpha. Our results show that, although HIF2 alpha was widely transcribed by kidney endothelium and vascular smooth muscle, knockouts displayed no detectable deficits in vessel development or VEGF or Flk-1 expression.